Fig. 6
Human PDGFRα+ periosteal progenitors have high osteogenic potential. a Representative histologic appearance of the human periosteum observed by H&E staining. The dashed line indicates the limit between the inner and outer layers of the periosteum. The dashed box indicates the location of b. b PDGFRα immunohistochemistry (shown in red) in human diaphyseal periosteum (n = 3). Nuclei, DAPI (blue). c PDGFRα− and PDGFRα+ cell isolation from the human diaphyseal periosteum. PDGFRα− and PDGFRα+ cells were isolated from the CD31−CD45− nonendothelial/nonhematopoietic cell population. Isotype control staining is shown. d Progenitor cell markers among human PDGFRα− and PDGFRα+ stromal cells from the same patient sample, including leptin receptor (LEPR) and Nestin (NES), were detected by qRT–PCR. e Cellular proliferation among human PDGFRα− and PDGFRα+ periosteal cells at 72 h as assessed by the MTS assay. f Osteogenic differentiation of PDGFRα− and PDGFRα+ periosteal cells as assessed by Alizarin Red (AR) staining. g Fibroblast colony-forming units (CFU-Fs) among human PDGFRα− and PDGFRα+ periosteal cells. h Schematic of the ossicle assay. PDGFRα− or PDGFRα+ cells (3 × 106) were intramuscularly implanted into NOD SCID mice using a DBX carrier and harvested 8-week later. i Micro-CT reconstructions of the implants. j Quantitative analysis of the micro-CT images of human PDGFRα− and PDGFRα+ stromal cells. The analysis included the bone volume (BV) and fractional bone volume (BV/TV). k Representative histologic appearance determined by H&E staining (left) and quantification of the bone area/tissue area (right). l Immunofluorescence staining of osteocalcin (OCN, red) and human-specific nuclei (green) within implants laden with PDGFRα− or PDGFRα+ cells. The white dashed lines demarcated the edges of the scaffold. m Representative ALP staining. Scale bars: 50 μm. B bone, BM bone marrow, PO periosteum. The dot plots represent an individual sample or animal, and the whisker plots indicate the mean and one-SD values. The experiments were performed in triplicate. n = 4 animals per group. *P < 0.05, **P < 0.01 and ***P < 0.001, as assessed by two-tailed Student’s t tests
